Our objectives are to purify and characterze certain chemical and biological properties of crotalase, a thrombin-like enzyme from eastern diamondback rattlesnake (Crotalus adamanteus) venom. We will also study the action of crotalase of blood coagulation factors, and its potential application in burn injury and tumor metastasis. The enzyme will be purified by combination of agamantine- or benzamidine-Sepharose affinity chromatography, and gel filtration or ion exchange chromatography. The amino acid sequence of crotalase will be determined as a subcontract to this application (see attachment). Certain amino acids known from previous work to be involved in the binding or active site of crotalase (serine, histidine and tyrosine) will be identified and their position in the amino acid sequence determined by chemical modification using site specific reagents. The physiological activities of crotalase on specific blood coagulation proteins will be investigated in detail. This will involve purification of these factors (specifically fibrinogen and factors V, VII, and VIII) and identification of peptide fragments by SDS-polyacrylamide gel electrophoresis after incubation with crotalase. Finally, since there are reports linking fibrin deposition to pathological processes in both burn injury and tumor metastasis and since crotalase is known to defibrinogenate animals in a benign fashion, the effect of crotalase induced defibrinogenation on the burn repair process and tumor metatasis will be evaluated using appropriate animal model systems.